Tuberculosis (TB) is a major cause of morbidity and mortality throughout the world. It is estimated that nearly 1% of the worlds population is newly infected each year and that approximately ⅓ of the worlds population is latently infected with Mycobacterium Tuberculosis tuberculosis (M. tuberculosis) the microorganism that causes the disease in man.
Immunocompetent individuals infected with M. tuberculosis in general have a lifetime risk of 10% for developing active TB, this risk increases many times if the individual is co infected with HIV. If left untreated each person with active pulmonary TB will infect 10 to 15 people each year [WHO, 2000]. For these reasons it is important to be able to detect TB infected individuals at an early stage of infection, to prevent the progression to active contagious TB (prophylactic treatment) or to treat the TB disease at an early stage. Therefore a fast and accurate diagnosis of M. tuberculosis infection is an important element of global health measures to control the disease.
Current diagnostic assays to determine M. tuberculosis infection include: culture, microscopy and PCR of relevant patient material, chest X-rays and the standard tuberculin skin test (TST). The three first methods are based on the identification of the M. tuberculosis bacteria and therefore depend on presence of bacteria in the sample. This demands a certain bacterial load and access to the infection site, and is therefore not suitable in early diagnosis. Chest X-ray is insensitive and only applicable in tuberculosis of the lung and in a progressed stage.
The standard tuberculin skin test, displaying a delayed type hypersensitivity reaction (DTH) is a simple and inexpensive assay, based on immunological recognition of mycobacterial antigens in exposed individuals. However it is far from ideal in detecting M. tuberculosis infection. It employs intradermal injection of purified protein derivative (PPD) which is a crude and poorly defined mixture of mycobacterial antigens some of which are shared with proteins from the vaccine sub-strain M. bovis bacille Calmette-Guèrin (BCG) and from non-tuberculosis environmental mycobacteria. This broad cross-reactivity of PPD causes the poor specificity of the TST, leading to a situation where BCG vaccination and exposure to non-tuberculosis mycobacteria gives a test result similar to that seen in a M. tuberculosis infected individual. The same concern applies for PPD when used in a blood cell based test. It is this immunological detection of M. tuberculosis infection our invention will improve.
M. tuberculosis infection mediates a strong cell mediated immune response and detection of T cells that are specific for this bacterium would be a suitable method to detect infection [Andersen, 2000].
To make a sensitive and specific cell mediated immunologically (CMI) based diagnostic reagent for M. tuberculosis infection two criteria needs to be meet to improve the accuracy:                The reagent should be broadly recognized by M. tuberculosis infected persons        The reagent should be specific for the tuberculosis bacteria, discriminating between TB infection and vaccination with the attenuated BCG strain or exposure to non pathogenic environmental mycobacteria.        
A highly specific reagent candidate should therefore be sought among antigens from the RD regions (regions of deletion) of the M. tuberculosis genome. These regions represent genomic deletions from the M. bovis BCG vaccine strain compared to the virulent M. tuberculosis strain [Behr, 1999]. Therefore, in theory, antigens from these regions would be excellent candidates for a TB diagnostic, i.e. they should not be recognized by healthy uninfected persons independent on their BCG vaccine status or exposure to non-pathogenic mycobacterial strains.
However out of all the predicted genomic ORF's (open reading frames) deleted from BCG it is not known per se which ones are in fact expressed into proteins and furthermore the immunoreactivity remains unknown until tested with sensitised lymphocytes from M. tuberculosis infected individuals either in a whole blood assay or on PBMC.
In our laboratory we have screened a large proportion of the deleted ORF's and only 10-15 of them were immunoreactive.
The diagnostic potential of the CFP 10 antigen (Rv3874), and The Esat 6 antigen (Rv 3875) two low molecular proteins from the RD 1 region, in a CMI based test is already well known [Arend, 2000; Arend 2001a; Arend 2001b; Brock, 2001; Lalvani, 2001a; Lalvani, 2001b; Lein, 1999; Munk, 2001; Ravn, 1999; Ulrichs, 2000; van Pinxteren, 2000; Vordermeier, 2001], also Rv1980 is well known as a CMI diagnostic antigen and in skin-test based tests. CFP 10 and Esat 6 proteins are very specific but each one used alone gives a sensitivity of about 75% which on its own is too low for a reliable diagnostic. Therefore combinations with other M. tuberculosis specific antigens needs to be found to broaden the recognition and thereby give a higher sensitivity without compromising the specificity.
Consequently there is a great need for a specific diagnostic reagent, comprising a cocktail of antigens or fusion polypeptides, which can be used either in vivo or in vitro to detect M. tuberculosis infections in humans and animals, and discriminate between TB infection and vaccination with the attenuated BCG strain or exposure to non pathogenic environmental mycobacteria.
We hereby present a cell mediated immunological (CMI) TB diagnostic based on a combination of epitopes from regions of the M. tuberculosis genome that are not present in the BCG vaccine strain or in non-tuberculosis mycobacteria. This diagnostic test has the advantage of being specific for M. tuberculosis infection, not recognizing BCG vaccinated individuals or individuals exposed to non-pathogenic mycobacteria. Furthermore the immunological test is not dependent upon the presence of M. tuberculosis bacteria in the sample and the test can be applied in any clinical form of infection with a high sensitivity.